PROJECT SUMMARY The human immune response against influenza is dominated by the production of hemagglutinin (HA)-specific antibodies (Abs). The yearly mutation rate in influenza HA proteins is 1-2% leading to development of new viral strains that are resistant to previous immunity. The other most predominant glycoprotein on the virion surface is neuraminidase (NA). Although immunogenic, predominance of human NA-specific Abs is much lower than HA, probably because NA expression is only one fourth the amount of HA on the virion surface. The yearly rate of mutation of NA is about half that of HA while part of the enzymatic site remains conserved across type A (IAV) and B (IBV) influenza viruses, making NA a potentially effective target for universal vaccine and therapeutic human monoclonal Ab (hmAb) development. Antibody responses targeting NA have demonstrated protective and therapeutic activity against influenza infection in animals, and in humans NA-inhibiting serum Abs have been correlated with effective protection, reduced disease severity, and duration of viral shedding, independent of or more strongly than HA-specific Ab responses, substantiating NA as a valuable target for the prevention and treatment of influenza in humans. Although seasonal human inactivated influenza vaccines (IIV) contain NA, the extent and mechanisms of action of protective human NA-specific humoral responses induced by vaccination are poorly resolved. Our research has demonstrated that IIV in humans does induce both IAV and IBV NA- specific B cells, and that the Ab clonal lineages they encode for include those that have broad and potent ability to protect and treat influenza infection. Further, we have demonstrated that these protective NA-specific B cell clonal lineages are present in long-lived bone marrow plasma cells in humans following IIV and are the likely source for their sustained presence in circulation. We posit that NA-mediated universal humoral protection, like HA, is dependent on B cell receptor/Ab specificity, however, to a greater extent than HA is also highly dependent on the precise Fc/IgG subclass composition of the NA-specific Ab repertoire. Our central hypothesis is that human IIV induces NA-specific B cell responses with broad protective potential, however, those with both the proper specificity and anti-viral activity to confer universal protection are subdominant and sporadically induced, hence at insufficient abundance to confer optimal protection. Through precisely defining the dynamics of those protective human NA-specific B cell clonal lineages, including their induction, frequency, persistence, precise specificity, and mechanisms of action, we expect to obtain insight on how to optimally stimulate them for future human universal vaccine strategies. To that end, we propose 1) define the evolution of human NA-specific B cell clonal lineages in response to seasonal IIV 2) assess the ability of IIV-induced human NA B cell lineages to inhibit infection and transmission and 3) evaluate the Fc-dependence of NA-specific hmAb protection. The process for the development of protective human NA-specific B cell clonal lineages will be defined and strategies to translate this to universal human vaccine-mediated protection from influenza infection identified.